Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

. Biochemical and cell-based analyses of 6-position modification with a halogen atom on lenalidomide. a, In vitro binding assay using recombinant proteins. Complex formation between biotinylated CRBN and FLAG-GST-SALL4 or IKZF1 in the presence of pomalidomide (Po), lenalidomide (Le), or 6-positionmodified Le (6-fluoro, F-Le; 6-chloro, Cl-Le; 6-bromo, Br-Le) was analysed using immunoblotting after a streptavidin pull-down assay. The experiment was repeated twice independently, with similar results. b, Immunoblot analysis of dose-dependent neosubstrate degradation in MCF7 cells. MCF7 cells were treated with DMSO, Po, Le, F-Le, Cl-Le, or Br-Le for 24 h, and the protein expression levels of neosubstrates were analysed using immunoblotting. The experiment was independently repeated thrice, with similar results. Source data are provided as a Source data file. Le, F-Le, Cl-Le, or Br-Le for nine days, and cell viability was analysed using the CellTiter-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 4). d, Dose-dependent anti-proliferative effect of lenalidomide derivatives on 5q MDS cell lines. MDS-L cells were treated with DMSO, Po, Le, F-Le, Cl-Le, or Br-Le for 20 days, and cell viability was analysed using the CellTiter-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 4). Source data are provided as a Source data file.  35 , and CK1α-CRBN complexes (PDB ID: 5FQD) 33 , respectively, which were determined using X-ray crystallography. b, Docking models of lenalidomide (Le) and five 6-position-modified Le (6-fluoro, F-Le; 6-chloro, Cl-Le; 6-bromo, Br-Le; 6-trifluoromethyl, F3C-Le; and 6trifluoromethoxy, F3CO-Le). Each compound and the residues located around the 6position of the phthalimide ring are shown in the stick model. These residues were set to       cell lines. MM1.S and H929 cells were treated with DMSO, Po, Le, F-P, Cl-P or F3C-Le for five days, and cell viability was analysed using the CellTiter-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 3). b, Anti-proliferative effect of lenalidomide derivatives on IMR32 cells. IMR32 cells were treated with DMSO, Po, Le, F-P, Cl-P or F3C-Le for five days, and cell viability was analysed using the CellTiter-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 3). c, Immunoblot analysis of BET proteins in pluripotent human embryonal carcinoma. NTERA-2 cells were treated with DMSO, Po-P, Le-P, F-P, Cl-P or F3C-P for 24 h, and the expression levels of BET proteins were analysed by immunoblotting. The experiment was independently repeated thrice, with similar results. d, Dose-dependent anti-    proliferative effects of PROTACs based on 6-position-modified lenalidomides on pluripotent human embryonal carcinoma. NTERA-2 cells were treated with DMSO, Po-P, Le-P, F-P, Cl-P or F3C-P for three days, and cell viability was analysed using the Cell Titer-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 3). e-f, Anti-proliferative effects of 6-positionmodified lenalidomides on NTERA-2 and HCT116 cells. (e) NTERA-2 or (f) HCT116 cells were treated with DMSO, Po, Le or 6-position-modified lenalidomides for three days (NTERA-2 cells) or two days (HCT116 cells), and cell viability was analysed using the CellTiter-Glo assay kit. Cell viability was expressed as the luminescence signal relative to the luminescence signal of DMSO, which was considered 100. Error bars denote standard deviation (biological replicates; n = 3). Source data are provided as a Source data file.

General information
All reactions were performed in oven-dried glassware under N2 atmosphere unless otherwise mentioned.
Solvents were transferred via syringe and were introduced into the reaction vessels. All the reactions were
The reaction was quenched with water, and Na2S2O3 aq. The reaction mixture was filtered through a pad of celite and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the title product as a brown oil (7.19 g, 89% yield).
The reaction mixture was cooled to room temperature, filtered through a pad of celite and acidified to pH 1-2 with 12 mol/L HCl aq. The resulting precipitate was filtered and dried in a vacuum to give the title product as a white solid (34 mg, 17% yield).

5-fluoro-2-methyl-3-nitrobenzoic acid
To a solution of 5-Fluoro-2-methylbenzoic acid (5.0 g, 32.4 mmol) in conc. H2SO4 (39 mL) was added fuming HNO3 (6.9 mL) dropwise slowly at 0 °C. The resulting mixture was stirred for 5 h at 0 °C. The reaction mixture was poured into crushed ice, and the resulting precipitate was filtered and washed with water. The precipitate was dried in a vacuum to give the title product as a white solid (3.8 g, 60% yield).

5-fluoro-3-nitrophthalic acid
A test tube was charged with 5-fluoro-2-methyl-3-nitrobenzoic acid (500 mg, 2.51 mmol), NaOH (301 mg, 7.53 mmol) in water (5.0 mL). The resulting mixture was heated to reflux and stirred for 1 h. The reaction mixture was cooled to room temperature, added KMnO4 (1.58 g, 10.0 mmol), heated to reflux, and stirred for 4 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite and acidified to pH 1-2 with 12 mol/L HCl aq. The resulting precipitate was filtered and dried in a vacuum to give the title product as a white solid (181 mg, 31% yield).

6-Fluoro-2-methyl-3-nitrobenzoic acid
To a solution of 2-fluoro-6-methylbenzoic acid (4.91 g, 32 mmol) in conc. H2SO4 (41 mL) was added fuming HNO3 (1.8 mL) dropwise slowly at 0 °C. The resulting mixture was stirred at 0 °C for 5 h. The reaction mixture was poured into crushed ice, and the resulting precipitate was filtered and washed with water. The precipitate was dried in a vacuum to give the title product as a white solid (3.92 g, 62% yield).
The residue was recrystallized with EtOH to give the title product as a off-white solid (330 mg, 87% yield).
The precipitate was dried in a vacuum desiccator to give the title product as a white solid (6.03 g, 83% yield).

Methyl 6-bromo-2-fluoro-3-methoxybenzoate
A 100 mL round bottom flask equipped with a deen-stark tube and condenser was charged with 6-bromo-2-fluoro-3-methoxy benzoic acid (5.73 g, 23.0 mmol), MeOH (23 mL), and conc. H2SO4 (1.0 mL). The resulting mixture was heated to reflux and stirred for 24 h. The reaction mixture was cooled to room temperature, concentrated, filtered, and washed with water. The resulting precipitate was dried in a vacuum desiccator to give the title product as a white solid (3.23 g, 55% yield).

5-Chloro-3-nitrophthalic acid
To a solution of 5-Chloro-2-methyl-3-nitrobenzoic acid (1.41 g, 6.5 mmol), NaOH (710 mg, 17.8 mmol) in water (13 mL) was added KMnO4 (8.25 g, 52.2 mmol). The resulting mixture was heated to reflux and stirred for 5 h. The reaction mixture was cooled to room temperature, added KMnO4 (8.25 g, 52.2 mmol), heated to reflux, and stirred for 6 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and acidified to pH 1-2 with 12 mol/L HCl aq. The resulting precipitate was filtered and dried in a vacuum to give the title product as a white solid (1.3 g, 81% yield).
The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give the title product a yellow solid (66.5 mg, 73% yield).

methyl 5-chloro-2-methyl-3-nitrobenzoate
To a solution of 5-chloro-2-methyl-3-nitrobenzoic acid (1 g, 4.6 mmol) in MeOH (46 mL) was added SOCl2 (1.0 mL, 13.9 mmol) dropwise slowly. The resulting mixture was heated to reflux and stirred for 14 h. The reaction mixture was cooled to room temperature and added water. The resulting precipitate was filtered and dried in a vacuum desiccator to give the title as a white solid (836 mg, 79% yield).
The resulting mixture was heated to reflux and stirred for 14 h. The reaction mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. The resulting precipitate was purified by flash column chromatography (Hexane/EtOAc = 95/5 to 9/1) to give the title product as a yellow solid (433 mg, 54% yield).

5-Bromo-3-nitrophthalic acid
To a solution of 5-bromo-2-methyl-3-nitrobenzoic acid (7.09 g, 26.9 mmol), NaOH (4.31 g, 108 mmol) in water (104 mL) was added KMnO4 (34.0 g, 215 mmol). The resulting mixture was heated to reflux and stirred for 1 h. The reaction mixture was cooled to room temperature, added KMnO4 (8.50 g, 215 mmol), heated to reflux, and stirred for 1 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and acidified to pH 1-2 with 12 mol/L HCl aq. The resulting precipitate was filtered and dried in a vacuum to give the title product as a white solid (2.33 g, 29% yield).
The resulting precipitate was filtered, washed with water, and dried in a vacuum desiccator to give the title product as a white solid (1.00 g, 71% yield).

2-methyl-3-nitro-5-(trifluoromethyl)benzoic acid
To a solution of 2-methyl-5-(trifluoromethyl)benzoic acid (1.0 g, 4.9 mmol) in conc. H2SO4 (10 mL) was added fuming HNO3 (1 mL) dropwise slowly at 0 °C. The resulting mixture was stirred for 2 h at room temperature. The reaction mixture was poured into crushed ice, and the resulting precipitate was filtered and washed with water. The precipitate was dried in a vacuum to give the title product as a white solid (1.2 g, 96% yield).

methyl 2-methyl-3-nitro-5-(trifluoromethyl)benzoate
To a solution of 2-methyl-3-nitro-5-(trifluoromethyl)benzoic acid (1.2 g, 4.9 mmol) in MeOH (25 mL) was added SOCl2 (1.1 mL, 15 mmol) dropwise slowly. The resulting mixture was heated to reflux and stirred for 12 h. The reaction mixture was cooled to room temperature and added NaHCO3 aq. Then the solvent was removed under reduced pressure and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The resulting mixture was purified by flash column chromatography (Hexane/EtOAc = 4/1) to give the title product as a colorless oil (1.08 g, 84% yield).